Ion pumps are important in maintaining cellular integrity. However, the molecular mechanisms of these pumps are not understood. Bacteriorhodopsin (bR) is a light-activated proton pump that is both abundant and stable. This relatively small and simple protein provides a convenient system for elucidating the mechanism of an active transport system. The same conformational change that occurs between the M, and N states of WT-bR has been observed in the photocycle of the high pH form of the D85N mutant. This mutant has a high pH form (D85Nalk) which pumps protons in the same direction as WT-bR. However, the Schiff base at the start of the photocycle in D85Nalk is deprotonated and resembles the M-state of WT-bR. The fact that the mutant undergoes a similar conformational change during its photocycle, even though its initial chromophore is deprotonated, suggests that the protonation state of the Schiff base is not critical to this conformational change. Studies of this mutant will contribute to a better understanding of the nature of this protein conformational change and its role in the proton-gating mechanism. [unreadable]We have used 15 N CPMAS experiments of L-"NI-Lys D85N to determine the protonation state of the Schiff Base in the neutral and alkaline states of this protein. Like Y-IT-bR, the dark adapted state of neutral D85N consists of both a 13-cis and an all-trans protonated SB species, as well as a small contribution from a 9-cis species. The high pH form of [_-"NI-Lys D85N shows a single peak at 286 ppm. The study of model Schiff bases showed that the unprotonated SB is downshifted by 145 ppm. This extreme downfield shift was also observed in the deprotonated M-state of WT-bR. Therefore, the peak at 286 pprn in the pH 10.8 D85N sample is assigned to the M-like state, which is the starting species of the D85Nalk photocycle. 13C CPMAS spectra of [ 12-"Cl-retinal D85N for both pH conditions were taken to determine the retinal isornerization state. The pH 6.5 spectrum shows a multiplet of resonances between 134 ppm and 122 ppm. These are attributed to the [ 12- "Cl-retinal. The peak at 134 ppm represents retinal in the alltrans isomerization state, while the peak at 122 ppm is the resonance for 13-cis retinal. This difference in chemical shift is due to the g-effect (strong steric interaction) between the protons on C12 and C15 of the retinal. Thus, the dark adapted state of neutral D85N consists of 13-cis and all-trans SB species, presumably due to an equilibrium of M-like, N-like and O-like states. The pH 10.8 spectrum has a sharp peak at 122 ppm. This represents a single, 13-cis, deprotonated Schiff Base observed in the M-like state.